A Xiphophorus two-gene, spontaneous melanoma model has been established, in which an oncogene known as mrk-2, and an uncharacterized tumor suppressor known as DIFF are implicated in the causation of melanoma. Recently a fish gene named CDKN2X has been cloned and is an excellent candidate for being the classically-defined DIFF tumor suppressor. The experimental goals of this candidate for being the classically-defined DIFF tumor suppressor. The experimental goals of this proposal serve to address key questions about the role of CDKN2X in the Xiphophorus melanoma model. The specific aims of this proposal are: 1. To study the zygotic status of CDKN2X in Xiphophorus melanomas, examining specific melanotic cell sub-types within tumors for loss of gene-copy phenomena. 2. To fully assess RNA expression characteristics of CDKN2X in numerous Xiphophorus control tissues and melanomas, using quantitative RT-PCR techniques. 3. To examine if DNA methylation within 5' and 3' CDKN2X CpG islands is involved in transcriptional repression. The potential role of DNA methylation acting on the CDKN2X gene will be assessed by initially comparing restriction fragment-length marker (RFLM) information obtained from methylation sensitive and insensitive restriction enzymes within CpG dinucleotide-rich areas known as CpG islands, and correlating these results with RNA expression of CDKN2X. These specific aims will serve to greatly enhance our understanding of the role of CDKN2X in melanoma formation.